Project Summary/Abstract - The long-term goals of the proposed research program are to provide insight into the complex dialogue that occurs each generation between humans and their microbiota during the critical period following birth. Recent research has indicated a strong correlation between outcomes of these early events and life-long health. However, the inaccessibility of colonized tissues and high diversity of the microbiota renders an in-depth study of early colonization of human tissues extremely challenging. When faced with such complex phenomena, biologists often turn to simpler model systems to provide insights into evolutionarily conserved features and reveal basic principles. To decipher the cellular and molecular mechanisms underlying the initiation of bacterial associations with apical surfaces of mucosal epithelia, the proposed program exploits the binary symbiosis between the bacterium Vibrio fischeri and its squid host, Euprymna scolopes. This relatively simple association has been studied for over two decades as a model for the chronic colonization of mucosa by Gram-negative bacteria. As in humans, the squid-vibrio association begins anew each generation, requiring a `winnowing' of other environmental bacteria that results in persistent association restricted to the coevolved partners. In this system, the process of symbiosis initiation occurs across ~100 microns over minutes to hours. It can be directly imaged in its entirety using confocal microscopy, which offers the rare opportunity to define, with high temporal and spatial resolution, the reciprocal molecular and biochemical dialogue that results in the establishment of a specific, life-long beneficial symbiosis. This project brings together two collaborators, each with expertise in the biology of one of the symbiotic partners, and introduces new technology to the study of host-microbe interactions, including: Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS), which allows precision tracking of symbiont molecules into host tissues; Hybridization-Chain-Reaction Fluorescent In Situ Hybridization (HCR-FISH), which enables visualization of rare transcripts in host and symbiont cells; NanoString, a new technology for simultaneous analysis of dozens to hundreds of targeted transcripts; and high-efficiency RNAseq, which produces robust transcriptional libraries from as little as 10 ng total RNA (~105 bacteria). Specific aims to be addressed are: (1) the examination of how symbiotic bacterial strain variation affects symbiosis onset and persistence; (2) the characterization of outer membrane vesicle (OMV) contents, their trafficking into host cells, and host responses to OMV cargo; and, (3) the investigation of the roles of vibrio virulence determinants in a non-pathogenic association. An understanding of the human microbiome is in its infancy, and this frontier field is currently at the stage of building paradigms. Within this context, as the squid-vibrio system has in the past, the results of the current study will shed light upon fundamental principles governing the onset of both beneficial and pathogenic associations.